INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

Pseudomonas aeruginosa is an opportunistic human pathogen that causes severe morbidity and mortality in patients with burns, cystic fibrosis, pneumonia, urinary tract infections, skin infections, cancer, acquired immunodeficiency syndrome, and ocular disease (1-11). P. aeruginosa is the leading cause of nosocomial pneumonia, with mortality as high as 70% and is the most common pathogen associated with intensive care unit infections (12, 13). Pseudomonas is the causative agent in more than 90% of loss of respiratory function in cystic fibrosis (14). Keratitis caused by P. aeruginosa is the most rapidly devastating bacterial infection of the human eye and the most common infection of the cornea among contact lens users in the United States (5, 15, 16). The administration of proper antibiotic therapy kills the bacteria but does not prevent intense pain and irreversible tissue damage.

Proteases are important in tissue damage caused by P. aeruginosa infection. P. aeruginosa secretes alkaline protease and two elastases (A and B) that have been characterized as exoenzymes and virulence factors (17-23). Invasiveness of P. aeruginosa in burn patients correlates with elastase production (22). Pseudomonas alkaline protease and elastase are produced in the lungs of patients with cystic fibrosis and have been shown to damage respiratory epithelium (23).

Protease IV is an uncharacterized exoenzyme that is regulated in a manner distinct from the other Pseudomonas proteases (24). We have recently determined that protease IV contributes significantly to corneal virulence (25, 26); however, characteristics of purified enzyme have not been previously reported. The current study focuses on the biochemical and enzymatic characterization of purified protease IV. The results, particularly the inhibitor studies, are significant because protease IV is resistant to the protease inhibitors currently available for chemotherapy of Pseudomonas infections.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Bacteria-- PA103-29 was provided by Dr. Barbara H. Iglewski, University of Rochester (Rochester, NY). PA103-29, like its parent (PA103), is deficient in elastase and alkaline protease, but unlike PA103, is also deficient in exotoxin A (19, 27, 28).

Analysis of Protease IV Activity-- Protease IV activity was assayed by its ability to cleave the chromogenic substrate, Chromozym PL (Boehringer Mannheim Biochemica, Indianapolis, IN), as described previously (25).

Localization of Active Protease IV-- To determine if active protease IV is sequestered inside the bacterial cell or periplasmic space, PA103-29 was grown in tryptic soy broth (Difco) at 37 °C for 24 h. The cells were pelleted by centrifugation at 8,000 × g for 15 min and washed in Tris buffer (0.01 M, pH 7.8). The periplasmic components were released by osmotic shock and tested for protease IV activity. Osmotic shock was accomplished as follows: cells were suspended in a sucrose buffer (20% sucrose, 0.003 M EDTA, 0.033 M Tris, pH 7.8), stirred for 20 min at room temperature, centrifuged to a pellet, resuspended in cold MgCl₂ (0.0005 M), and stirred at 4 °C. The osmotically shocked cells were centrifuged, and the resulting supernatant was assayed for protease IV activity.

Purification of Protease IV-- Protease IV was purified from concentrated culture supernatants of P. aeruginosa strain PA103-29 using ion exchange and molecular sieve chromatography (Table I). Cultures (46 liters per batch) of strain PA103-29 were grown for 24 h at 37 °C in M-9 medium (29), supplemented with monosodium glutamate (0.13 M) and glycerol (0.1 M), and centrifuged at 8,000 × g for 15 min to pellet the cells. The supernatant was filtered through a 0.45-µm capsule filter (Versiflow; Fisher) to remove any remaining bacteria. Sodium azide was added to a concentration of 0.02%, and the culture supernatant was concentrated to approximately 80 ml by a two-step ultrafiltration process using first a spiral membrane concentrator and then a stirred cell concentrator, both with a 10,000 molecular weight cutoff filter (Amicon Inc., Beverly, MA). The concentrated supernatant was then dialyzed against 10 mM ammonium acetate buffer. The dialyzed supernatant was applied to a carboxymethyl-cation exchange column (5 × 30 cm; Bio-Rad). The column was washed with 10 mM ammonium acetate (pH 6.8, 0.02% sodium azide) and eluted with a gradient formed by the addition of 10 mM ammonium acetate buffer at pH 9 to 10 mM ammonium acetate buffer at pH 6.8. Fractions with high protease IV activity were pooled, concentrated, and applied to a Sephacryl 300 column (2.5 × 50 cm; Pharmacia Biotech, Uppsala, Sweden) that had a 33-ml void volume. Fractions (2.0 ml) were eluted with 10 mM Tris buffer, pH 7.0, containing 0.02% sodium azide. Fractions with protease IV activity were pooled and concentrated. Total protein from each step of the purification was determined using the bicinchoninic acid assay (BCA; Sigma). Purity of the protease was determined by electrophoresis on 12.5% SDS-PAGE¹ gels. Gels were silver-stained.

Analysis by Mass Spectrometry-- Purity was confirmed by mass spectroscopy using a Voyager-DE^TM Biospectrometry^TM Workstation (Perspective Biosystems, Inc., Framingham, MA). Protease IV (20 µg/ml) was dialyzed against sterile deionized water using a Microcon concentrator (10,000 M_r cutoff, Amicon, Beverly, MA). Protease IV (1 µl) was mixed with 9 µl of sinapinic acid matrix solution (10 mg of sinapinic acid, 567 µl of H₂O, 100 µl of 1% trifluoroacetic acid, and 333 µl of acetonitrile). An aliquot of sample was loaded into the mass spectrometer. The sample was analyzed using an accelerating voltage of 20,000, grid voltage of 94.5%, guide wire voltage of 0.04%, and a low mass gate of 400.0. Fifty-two scans were averaged to produce the final molecular weight.

N-terminal Sequence Determination-- N-terminal amino acid sequence analysis of the purified protease IV protein was obtained using the method of Moos et al. (30). Purified protease IV (50 µg) was electrophoresed (10 mA, 75 V) for 60 min on 12% SDS-PAGE in the presence of the tracking dye pyronin Y. Protein was transblotted (24 V for 1 h) from the polyacrylamide gel to a polyvinylidene difluoride membrane (Millipore, Bedford, MA) using CAPS buffer (10 mM), 10% methanol, pH 11). The polyvinylidene difluoride membrane was stained with Ponceau S (0.2% in 1.0% acetic acid), and the single protein band was excised and air-dried. N-terminal sequencing of 10 amino acids was carried out using a model 494 gas phase protein sequencer (Applied Biosystems, Foster City, CA) with an Applied Biosystems model 785 programmable absorbance detector. This procedure was repeated using three different preparations. The sequence of 10 amino acids was compared with other known peptide sequences using the BLAST network homology search service at the National Center for Biotechnology.

Isoelectric Focusing-- Purified protease IV (1 ml), dialyzed against water, was mixed with Rotolyte solutions (AMPSO 15 ml and triethanolamine 15 ml; Bio-Rad) and deionized water (30 ml). The sample was loaded into the Rotofor cell (Bio-Rad) and subjected to 15 watts constant power for 4 h at 4 °C. The pH and protease IV activity of the fractions were determined and aliquots were analyzed by SDS-PAGE (12.5%).

Analysis of Substrate Susceptibility to Protease IV-- Several chromogenic substrates were reacted with purified protease IV at room temperature (Table II). Reactions were started with the addition of protease IV and the change in optical density at 410 nm was determined every 5 min for 30 min. Negative controls consisted of heat-inactivated protease IV mixed with substrate. Protease IV (1 µg) was also mixed with 4 µg of fibrinogen (Sigma), plasminogen (Sigma), plasmin (Sigma), C3 complement component (Calbiochem-Novabiochem Corp., La Jolla, CA), C1q complement component (Calbiochem-Novabiochem), or immunoglobulin G (Calbiochem-Novabiochem) in 50 mM Tris, pH 8.2. Controls consisted of each substrate without protease IV or protease IV alone. Reaction mixtures were incubated for 1 h at 37 °C. The protease was inactivated by the addition of an equal volume of SDS-PAGE sample buffer containing 300 mM 2-mercaptoethanol, followed immediately by boiling for 5 min. Degradation was determined by electrophoresis of the reaction mixtures on SDS-PAGE (12.5%) under reducing conditions.

Determination of Optimal pH for Protease IV Activity-- The reactions between purified protease IV (50 ng) and substrate were assayed in triplicate for 30 min in solutions ranging from pH 2 to 13. Controls consisted of the Chromozym PL or Val-Leu-Lys-4-nitroanilide substrates at the pH values tested without protease IV.

Determination of Optimal Temperature for Protease IV Activity-- A mixture of NaCl (0.9%), Tris buffer (50 mM, pH 8.2), and Chromozym PL (2 mg/ml) was incubated for 15 min at 65, 55, 45, 37, 25, or 10 °C. After the 15-min incubation, protease IV (50 ng at each temperature) was added to the substrate solutions, and the optical density at 410 nm was recorded for 30 min at that temperature. The ratio of substrate to enzyme was predetermined to ensure excess substrate throughout the assay. Controls consisted of the reaction mixture incubated at each temperature tested without the addition of protease IV.

Kinetic Analysis-- Kinetic assays were performed at pH 10 and at 25 °C using tosyl-Gly-Pro-Lys-paranitroanilide or Val-Leu-Lys-paranitroanilide as substrate. The V_max and K_m values were determined by plotting the substrate concentration versus the initial velocity of each reaction using the scientific graphing program Microcal 3.5 (Microcal Software, Inc., Northampton, MA).

Inhibitor Studies-- Preincubation of protease IV (50 µg in 10 µl of 50 mM Tris, pH 8.2) and inhibitor (80 µl in 50 mM Tris-HCl, pH 8.2, and 10 µl of NaCl, 150 mM) was carried out at room temperature for 30 min before the substrate (Chromozym PL) was added. Assays were performed in triplicate. Appropriate controls, in which the inhibitor was replaced with solvent, were assayed in parallel. The activity of each inhibitor used was confirmed by demonstrating its activity against susceptible enzymes (Table III).

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Osmotic shock or cell lysis of pelleted bacteria failed to liberate any protease IV activity; all detectable active protease IV was secreted into the culture medium during growth. Protease IV was purified from the concentrated culture supernatant fraction of P. aeruginosa strain PA103-29 by ion exchange and molecular sieve chromatography. Protease IV, unlike the majority of protein (~90%) in the culture supernatant, adhered to the cation exchange resin. Protease IV was eluted from the ion exchange column between pH 7.3 and 8.8 (Fig. 1A).

View larger version (39K): [in this window] [in a new window]   Fig. 1.   Purification of protease IV from concentrated culture supernatants of PA103-29. A, chromatography of PA103-29 concentrated culture supernatant on a carboxymethyl agarose (Bio-Rad). The activity of protease IV was detected using the Chromozym PL assay (). The majority of proteins (~90%) in the concentrated culture supernatant failed to bind to the column (). Protease IV eluted when the pH reached pH 7.8 (fractions 119-150) (×). B, molecular sieve chromatography of protease IV-rich fractions derived from ion exchange chromatography of PA103-29 concentrated culture supernatants. Protease IV eluted as a single peak with a molecular mass of approximately 30 kDa. C, SDS-PAGE analysis of protease IV-enriched fractions obtained during enzyme purification. Concentrated culture supernatant (lane 1), protease IV-rich fractions from cation exchange chromatography (lane 3), protease IV-rich fractions from molecular sieve chromatography (1.0 µg) (lane 2), and molecular mass standards (lane 4). Protease IV eluted from the molecular sieve column as a homogeneous band with an apparent molecular mass of 30 kDa. D, mass spectroscopy of protease IV reveals a single peak with a molecular mass of 26,383.9 Da. E, SDS-PAGE analysis of protease IV auto-degradation. Preparations of purified protease IV demonstrate a 17-kDa breakdown product (lane 1). Lane 2, molecular mass standards.

Protease IV was further purified using a size exclusion column (Sephacryl 300). Protease IV eluted from the Sephacryl 300 column as a single peak with a molecular mass of approximately 30 kDa (Fig. 1B). The stepwise purification of protease IV resulting in greater than a 500-fold increase in specific activity is summarized in Table I. Analysis by SDS-PAGE of the protease-rich peak from the Sephacryl column revealed a single protein band with a molecular mass of approximately 30 kDa (Fig. 1C). Protease IV demonstrated a single peak with a molecular mass of 26,383.9 Da by mass spectrometry (Fig. 1D).

The N-terminal amino acid sequence of protease IV was determined to be alanine-glycine-tyrosine-arginine-aspartic acid-glycine-phenylalanine-glycine-alanine-serine (AGYRDGFGAS). Analysis of this sequence using BLAST network homology sequence searches failed to reveal similarity with any previously described protein.

Upon storage of purified protease IV, there was a decrease in the amount of enzyme migrating as a 30-kDa protein on SDS-PAGE and the appearance of a band at approximately 17 kDa. N-terminal sequencing demonstrated that the 17-kDa degradation product had the same amino acid sequence as the 30-kDa protein (Fig. 1E). The total enzyme activity remained constant despite the apparent reduction in molecular weight.

The isoelectric point of protease IV was 8.7 (data not shown). Enzyme assays conducted at eight different pH values indicated that maximal protease IV activity occurred at pH 10 (Fig. 2A). Protease activity increased from pH 4 to 9; pH 11 demonstrated approximately 85% of maximal activity, but there was no activity at pH  12. 

View larger version (34K): [in this window] [in a new window]   Fig. 2.   The effect of pH and temperature on the activity of purified protease IV. A, purified enzyme was placed in buffers at various pH values ranging from pH 2 to 13. The substrate, Chromozym PL, was added, and the optical density (410 nm) was measured every 5 min for 30 min. At the termination of the experiment, the pH of each solution was confirmed. Maximum enzyme activity was at pH 10.0. Negative controls consisted of the substrate incubated with each buffer without the addition of protease IV (all assays were performed in triplicate). B, temperature for optimal protease IV activity. Protease IV was incubated for 30 min at various temperatures in triplicate, and the enzymatic activity was determined using Chromozym PL. The highest enzymatic activity was at 45 °C. C, degradation of biologically important proteins. Reaction mixtures (25 µl) incubated at 37 °C for 1 h contained protease IV (1 µg) and 4 µg of: fibrinogen (lane 3), plasminogen (lane 5), plasmin (lane 7), complement component C3 (lane 9), complement component C1q (lane 11), or IgG (lane 13). Controls included individual proteins incubated under identical conditions, including: fibrinogen (lane 2), plasminogen (lane 4), plasmin (lane 6), complement component C3 (lane 8), complement component C1q (lane 10), IgG (lane 12), and protease IV (lane 14). Low molecular mass standards were electrophoresed in lane 1. Degradation of proteins was determined by electrophoresing each reaction mixture or its control (12.5 µl) on SDS-PAGE (12%) under reducing conditions.

The enzymatic activity of protease IV increased with temperature from 10 to 45 °C with maximal activity occurring at 45 °C (Fig. 2B). Protease IV retained its enzymatic activity over 1 h at 45 °C (data not shown). Studies of the thermal liability of protease IV demonstrated that approximately 90% of the activity was abolished by incubation of the enzyme at 60 °C for 40 min or 80 °C for 1 min (data not shown).

Protease IV degraded fibrinogen, plasminogen, and C3 as determined by the complete disappearance of the relative bands present in the control reactions lacking protease IV (Fig. 2C). Plasmin, C1q, and IgG were partially degraded by protease IV, as evidenced by a decrease in the relative bands present in the control reactions concomitantly with the appearance of lower molecular weight bands in the reactions containing protease IV. Neither protease IV nor any substrate incubated alone demonstrated significant autodegradation under these conditions.

Of the chromogenic substrates analyzed for susceptibility to protease IV, only Chromozym PL (tosyl-Gly-Pro-Lys-4-nitroanilide) and Val-Leu-Lys-4-nitroanilide were digested, suggesting that this enzyme cleaves on the carboxyl side of lysine residues (Table II). Protease IV was not able to cleave substrates that terminated with arginine residues linked to the paranitroanilide or substrates with a single amino acid linked to paranitroanilide.

Kinetic analysis of the hydrolysis of tosyl-Gly-Pro-Lys-4-nitroanilide and Val-Leu-Lys-4-nitroanilide by protease IV was determined by plotting substrate concentration versus velocity (Fig. 3). The K_m and V_max for tosyl-Gly-Pro-Lys-paranitroanilide (Chromozym PL) were 319 µM and 1.33 µM/min, respectively. The K_m and V_max for Val-Leu-Lys-paranitroanilide were 727 µM and 0.74 µM/min, respectively.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Time course hydrolysis of tosyl-Gly-Pro-Lys-4-nitroanilide (Chromozym PL) () and Val-Leu-Lys-4-nitroanilide (). Km and Vmax values were calculated by plotting substrate concentration versus initial velocity. The reaction mixture contained 10 µl of 0.9% sodium chloride, 80 µl of 50 mM Tris, pH 10.0, 10 µl of protease IV (50 ng), and 20 µl of substrate. Substrate concentrations ranged from 0.025 to 4 mM and were run in triplicate. Optical density was measured every 2 min over a 2-h interval. The initial velocity at each substrate concentration was determined by calculating the change in optical density per min divided by the extinction coefficient (E = 9.75/mM × cm × 0.53).

Fifteen enzyme inhibitors were tested for their ability to block the hydrolysis of Chromozym PL. The only compound to demonstrate complete inhibition of protease IV activity was TLCK (Table III). Partial inhibition was observed with the serine protease inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. The reducing agents dithiothreitol (1.0 mM) and 2-mercaptoethanol (150 mM) also demonstrated complete inhibition of protease IV activity (Table III). EDTA at 50 mM caused a 30% inhibition of protease IV activity.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

Protease IV activity correlates with virulence and is known to contribute to the corneal damage that often results in a loss of visual acuity and even blindness following Pseudomonas keratitis (25, 26). We describe in this study the initial characterization of this enzyme. Protease IV is active only as an extracellular enzyme. Ultrafiltration followed by ion exchange and molecular sieve chromatography were effective in increasing the specific activity of protease IV by more than 500-fold and resulted in apparent homogeneity as evidenced by a single band on SDS-PAGE and a single peak of macromolecule in the mass spectrometer.

Elution of this enzyme in a single symmetrical peak on gel filtration and its mobility on SDS-PAGE under reducing conditions indicate that the protease IV monomer has an estimated molecular mass of approximately 30 kDa. Mass spectroscopy demonstrated that protease IV has a molecular mass of 26.3 kDa as a monomer. Protease IV aggregates when electrophoresed in the presence of SDS (1.0%) under nonreducing conditions in a zymogram (25). The aggregate was observed only on zymograms and is probably the result of partial denaturation of the enzyme after exposure to SDS during zymography.

Protease IV cleaves on the carboxyl side of lysine residues as determined by substrate susceptibility studies. The lack of cleavage of the substrate L-Lys-4-nitroanilide suggests that protease IV requires the presence of more than one amino acid for proteolysis to occur. Protease IV shares similar substrate affinity with another protease (Ps-1) of Pseudomonas (31), but because no information on the amino acid or nucleotide sequence of Ps-1 has been reported, the relatedness of the two enzymes remains to be confirmed.

Inhibition studies suggest that protease IV is a serine protease. Diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, which irreversibly and specifically react with active site serine residues (32, 33), partially inhibited protease IV activity. TLCK totally abolished the activity of protease IV, suggesting the presence of a histidine residue in the active site of the enzyme (34). TLCK can also react with active sulfhydryl groups and inhibit cysteine proteases; however, our inhibitor data overall suggest that protease IV is a serine protease. EDTA did inhibit the enzyme activity but not at the low concentrations inhibitory for Pseudomonas metalloproteinases. Furthermore, inhibition by dithiothreitol and 2-mercaptoethanol suggests that disulfide bonds could be important in maintaining the molecular conformation required for activity.

We have previously demonstrated that protease IV can restore corneal virulence to a mutant strain of P. aeruginosa that does not produce protease (26). The current research shows that protease IV is capable of degrading a variety of biologically important molecules associated with the innate and humoral immune responses to infection. These reactions could contribute to virulence by interfering with critical host defense mechanisms. Unknown as yet is the ability of protease IV to directly damage host structural proteins (i.e. collagen or proteoglycans) and/or activate host proteases present in the cornea. Such reactions could further explain the role of protease IV in corneal virulence.

Protease IV is distinct and unique from other Pseudomonas proteases associated with virulence (17-23, 28). Research is currently in progress to determine the therapeutic value of protease inhibitors that are reactive with protease IV in reducing tissue damage that occurs during Pseudomonas infections.